This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. ARGONAUTE1 protein is the key component of RNA Induced Silencing Complex (RISC), which is the catalytic protein machinery for small RNA guided gene silencing. Ago proteins bind to a variety of auxiliary protein factors in vivo to form holo-RISC to perform the function. Deficiency of these auxiliary factors significantly compromises RISC function. The overall goal is to perform structural analysis of holo-RISC in different status. Given the size of this holo-RISC (ranging from 1~2 MDa), electron cryo-microscopy turns out to be a preferred tool to reveal the structure of this complex. We will purify and identify miRNA holo-RISC from Arabidopsis thaliana and the analogous complex from Thermus thermophilus. We have monoclonal antibodie, which specifically recognize these holo-RISCs. We are testing protocols to obtain homogeneous holo-RISCs. We will address several fundamental and long standing questions in the field of RNA silencing: What factors constitute holo-RISC? How is holo-RISC assembled? Studies of the structure and assembly of holo-RISC will provide not only the structure by also the mechanistic understanding of RNAi. Such efforts will eventually facilitate rational design of new and improved gene-silencing technologies. Applications of RNA silencing technologies could have a significant impact on a broad spectrum of biomedical sciences, ranging from understanding the basic biology, uncovering the molecular basis of human disease, to developing novel therapeutics.